Lyme disease spirochetes disseminate from the site of inoculation via tick bite, and are able to persist despite the host immune response, indicating that interactions with mammalian cells occur continually during infection. B. burgdorferi binds to several integrins, which are critical to diverse cellular functions including maintenance of tissue integrity and participating in the immune response. We identified the B. burgdorferi surface protein P66 as a ligand for the 3-chain integrins. We generated B. burgdorferi p66- mutants, and showed that these mutants are not infectious in mice, but are in ticks. The p66 mutants are cultivable from the site of infection in mice for only 1-2 days, and are not infectious even when delivered by ticks. The p66- mutants do survive in the protected environment of dialysis membrane chambers implanted into the peritoneal cavity, and in the tick after the bloodmeal. Thus, the p66- mutants are not simply compromised in cell integrity or essential metabolic function. Complementation of the mutants by restoration of p66 to the chromosome restores infectivity in mice. P66 affects the responses of human cells in culture to the bacteria, and our results are consistent with the hypothesis that host responses to pathogens, such as phagocytosis and maintenance of cell shape and tissue integrity, are affected by P66. Our results are also consistent with a role for P66 in regulation of vascular permeability, and the presence of B. burgdorferi in ticks increases repletion weight, suggesting that increased vessel permeability in response to B. burgdorferi could potentially benefit the tick as well as the bacterium. Our goal is to determine how B. burgdorferi overcomes host barriers to the establishment of persistent, disseminated infection, which is critical to the life of the organism. Our hypothesis is that the integrin binding activity of P66 is essential to B. burgdorferi in the mammalian host due to manipulation of host cell functions such as phagocytosis and maintenance of tissue barriers. We will test our hypothesis using three approaches: Aim 1: Test the hypothesis that the p66- mutants are rapidly phagocytosed by cells of the innate immune system at the site of inoculation. We will use imaging and targeted cell ablation to determine whether P66 is required for B. burgdorferi to avoid phagocytosis by macrophages and dendritic cells at the site of inoculation. Aim 2: Test the hypothesis that the p66- mutants are defective in crossing into and out of the vasculature. We will use imaging to analyze and quantify B. burgdorferi interactions with the vasculature, and will quantify B. burgdorferi chemotaxis toward known attractants. Aim 3: Test the hypothesis that 3-chain binding by P66 is essential to the ability of B. burgdorferi to cause infection. In conjunction with Aims 1 and 2, reagents that block integrin function and bacteria expressing site- directed mutations in P66 will be used to investigate the mechanistic role of P66 in mammalian infection. These studies will define how B. burgdorferi overcomes host barriers to the establishment of infection, which is critical to the organism in nature, and may suggest novel routes to prevention and treatment of Lyme disease.